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Method for specifically detecting polypeptide drug based on elimination of interference of intermolecular disulfide bonds

A disulfide bond and intermolecular technology, applied in measuring devices, material separation, analysis materials, etc., can solve problems such as interference in peptide detection, improve sensitivity and accuracy, eliminate disulfide bond interference, and avoid interference

Active Publication Date: 2019-08-23
JIANGSU FOOD & PHARMA SCI COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, when detecting peptides, due to the existence of disulfide bonds, the disulfide bonds will also be reduced during the traditional biotin conversion process, which will interfere with the detection of peptides

Method used

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  • Method for specifically detecting polypeptide drug based on elimination of interference of intermolecular disulfide bonds

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Experimental program
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Embodiment 1

[0030] Materials and Reagents

[0031] In this embodiment, Exenatide reagent purchased from Fluka Company of Switzerland was used as the experimental reagent. The ethanol, salicylic acid and stearic imide used were purchased from Guangzhou Chemical Reagent Factory. Sodium polyethoxide and sodium bicarbonate used were purchased from Qingdao Reagent Factory. The HP 1100 high-performance liquid chromatograph purchased from HP Company and the UltraFlix purchased from Germany Bruker Company were adopted. TM III Matrix-assisted laser desorption time-of-flight mass spectrometer as detection and analysis instrument.

[0032] experimental method

[0033] It mainly includes the following steps 1: prepare the exenatide to be detected, and pretreat it with a salicylic acid solution containing 0.4% ethanol to adjust the solution to pH 5; step 2: add the solution containing 0.4% stearimide of diethyl benzoate to block the free sulfhydryl groups in exenatide; step 3: remove excess stear...

Embodiment 2

[0042] experimental method

[0043] It mainly includes the following steps 1: prepare the glutathione to be detected, and perform pretreatment with a salicylic acid solution containing 0.4% ethanol to adjust the solution to pH 6; step 2: add to the solution obtained in step 1 containing 0.4% stearimide of dimethyl benzoate to block free sulfhydryl groups in glutathione; step 3: remove excess stearimide in step 2 by adding acid, and then obtain Add 2 g sodium polyethoxide to the solution of exenatide to block the intermolecular disulfide bonds; Step 4: remove unbound blocking reagent, use 0.4% sodium carbonate to adjust the pH of the solution to 8, and then use sulfoxylate Sodium reduces the modified polypeptide cysteine ​​sulfhydryl group to a free sulfhydryl group; step 5: adding benzenesulfonic acid imide to the solution obtained in step 4 to label the free sulfhydryl group in glutathione; Glutathione labeled with the free sulfhydryl group was affinity purified and then ana...

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Abstract

The invention provides a method for specifically detecting a polypeptide drug based on elimination of interference of intermolecular disulfide bonds. The method comprises the following steps: adding imide stearate for blocking free sulfhydryls in a polypeptide sample, and then adding poly(sodium ethoxide) for blocking intermolecular disulfide bonds in the polypeptide, so that interference producedby the free sulfhydryls and the intermolecular disulfide bonds in a polypeptide detection process is avoided. The method provided by the invention is simple and effective and has high detection sensitivity and accuracy, thereby having good cost effectiveness.

Description

technical field [0001] The invention relates to the field of polypeptide drug detection, and more specifically relates to a method for specifically detecting polypeptides under the condition of excluding the interference of intermolecular disulfide bonds. Background technique [0002] Peptide drug is a special drug between traditional chemical drugs and macromolecular drugs, and it is one of the hot spots in drug development in recent years. It has the characteristics of low toxic and side effects, less dosage, strong biological activity and good curative effect. Now there are more than 60 synthetic peptide drugs on the market in the world, and there are more than 30 synthetic peptide drugs included in the domestic pharmacopoeia. The dosage forms of polypeptide drugs are mostly injections and injection powders, and there are also some new types of agents, such as injection microspheres, sustained release agents, and nasal sprays. [0003] In peptide synthesis, the disulfid...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 马宇春朱玉洁
Owner JIANGSU FOOD & PHARMA SCI COLLEGE